An Efficient Rapid System for Profiling the Cellular Activities of Molecular Libraries
Total Page：16
File Type：pdf, Size：1020Kb
An efficient rapid system for profiling the cellular activities of molecular libraries Jonathan S. Melnick*†, Jeff Janes†‡, Sungjoon Kim†‡, Jim Y. Chang‡, Daniel G. Sipes‡, Drew Gunderson‡, Laura Jarnes‡, Jason T. Matzen‡, Michael E. Garcia‡, Tami L. Hood‡, Ronak Beigi‡, Gang Xia‡, Richard A. Harig‡, Hayk Asatryan‡, S. Frank Yan‡, Yingyao Zhou‡, Xiang-Ju Gu‡, Alham Saadat‡, Vicki Zhou‡, Frederick J. King‡, Christopher M. Shaw‡, Andrew I. Su‡, Robert Downs‡, Nathanael S. Gray‡, Peter G. Schultz*§, Markus Warmuth‡§, and Jeremy S. Caldwell‡§ ‡Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121; and *The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Peter G. Schultz, December 29, 2005 Rapid quantitative methods for characterizing small molecules, of disorders, most notably cancer (4, 5). For example, spontaneous peptides, proteins, or RNAs in a broad array of cellular assays translocations in which tyrosine kinase domains become fused to would allow one to discover new biological activities associated other genes (such as Bcr, Tel, and NPM) have been identified as the with these molecules and also provide a more comprehensive etiological basis for multiple B cell lymphomas, including the profile of drug candidates early in the drug development process. Philadelphia chromosome Bcr-Abl and chronic myelogenous leu- Here we describe a robotic system, termed the automated com- kemia (6, 7). It remains a significant challenge to develop selective pound profiler, capable of both propagating a large number of cell inhibitors for tyrosine kinases given their homology and potential lines in parallel and assaying large collections of molecules simul- structural plasticity. To this end, we have established a panel of taneously against a matrix of cellular assays in a highly reproduc- tyrosine-kinase-dependent cellular assays by creating stably ex- ible manner. To illustrate its utility, we have characterized a set of pressed Tel-tyrosine kinase fusions representing each branch of the 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinase- tyrosine kinase phylogeny. A library of Ͼ1,000 kinase-directed dependent cellular assays in dose–response format in a single heterocycles was then profiled against this panel of cellular assays. experiment. Analysis of the resulting multidimensional dataset The resulting dataset was examined for global structure–activity CELL BIOLOGY revealed subclusters of both inhibitors and kinases with closely relationships (SARs), potency and selectivity correlations, and correlated activities. The approach also identified activities for the opportunistic side activities. p38 inhibitor BIRB796 and the dual src͞abl inhibitor BMS-354825 and exposed the expected side activities for Glivec͞STI571, includ- Results and Discussion ing cellular inhibition of c-kit and platelet-derived growth factor Construction of an ACP System. A robotic system was designed that receptor. This methodology provides a powerful tool for unravel- combines cell culture processes with compound screening capabil- ing the cellular biology and molecular pharmacology of both ities to create a massively parallel compound profiling system. This naturally occurring and synthetic chemical diversity. system mechanically integrates all steps involved in cell line main- tenance, including propagation (passaging, splitting, cell density, drug discovery ͉ high-throughput screening ͉ tyrosine kinase and viability determination) with the steps involved in performing compound screens (cell dispensing into microtiter plates, com- he ability to simultaneously interrogate the activities of a library pound addition, incubation, detection, reagent addition, and plate Tof molecules against a large panel of cellular assays would reading͞imaging), as described in Materials and Methods and as provide a rapid efficient means to begin to characterize and depicted in Fig. 1. The design specifications include the ability to correlate the biological properties of both natural and synthetic rapidly profile thousands of compounds in dose–response format in chemical diversity. For example, libraries of noncoding RNAs, miniaturized 1,536-well plate format against hundreds of roboti- mutant growth factors, small molecule kinase inhibitors, or even cally maintained cell-based assays in a highly reproducible way. The existing drugs could be assayed for their potency and selectivity in focus on cell maintenance required strict environmental control of pathway-based or receptor screens or toxicity and metabolic sta- humidity, temperature, sterility, and cell line cross-contamination, bility in diverse cell types to discover a new biological activity or completely distinct from those confronted using biochemical͞ optimize the pharmacological properties of a molecule (1–3). protein assays. The combination of automated tissue culture and Although whole-cell systems represent an attractive milieu to robotic assay technologies enables small-molecule screens to be characterize gene and small-molecule function, no robust and performed on an unprecedented scale in cell-based formats. This systematic method exists to correlate chemical structure and bio- system can also be adapted to screen other molecular libraries, logical activity across a large number of molecules and cellular including secreted peptides and proteins, antibodies, cDNAs, and assays. To address this problem, we have developed an approach siRNAs against collections of cellular assays targeting either spe- that affords rapid cost-effective broad-based cellular profiling in cific gene families (kinases, G-protein-coupled receptors, pro- parallel against molecular libraries. An industrial-scale automated teases, nuclear hormone receptors, etc.), signaling pathways (us- compound profiling (ACP) system has been designed, which con- ing reporter gene, phenotypic, and image-based readouts), or sists of an automated tissue culture system for propagating cell lines, pharmacological properties (metabolic stability, cellular toxicity, or integrated with a system for automatically performing miniaturized transport). cell-based assays in 384- or 1,536-well microplates. The ACP can rapidly test thousands of arrayed molecules, in replicates, in dose– response format against hundreds of unique cellular assays in a Conflict of interest statement: No conflicts declared. single experiment. Freely available online through the PNAS open access option. To demonstrate this capability, we focused on the problem of Abbreviations: ACP, automated compound profiling; SAR, structure–activity relationship. identifying selective small-molecule inhibitors of protein tyrosine †J.S.M., J.J., and S.K. contributed equally to this work. kinases. Tyrosine kinases play a key role in many cellular processes, §To whom correspondence may be addressed. E-mail: [email protected], mwarmuth@ including development, differentiation, and proliferation; misregu- gnf.org, or [email protected]. lation of tyrosine kinase expression and activity leads to a number © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0511292103 PNAS ͉ February 28, 2006 ͉ vol. 103 ͉ no. 9 ͉ 3153–3158 Downloaded by guest on October 1, 2021 cell line model has frequently been used to demonstrate the differential cytotoxicity of kinase inhibitors against transformed IL-3-independent cells versus parental Ba͞F3 cells grown in the presence of IL-3 (16–18). After selection for puromycin and IL-3 withdrawal, 35 of the 81 fusion kinase constructs gave rise to unique cell populations exhibiting growth factor independence. Fig. 2b summarizes the identity of the kinases found to induce growth factor independence in Ba͞F3 cells. All cells were further validated as described (Ma- terials and Methods) by Western blot and RT-PCR͞sequencing of the respective fusion and by using sets of reference compounds for each individual kinase. In the latter case, two independently gen- erated cultures of IL-3-independent cells were required to show similar responses to reference compounds. To determine whether kinase inhibitor activity was accurately reflected by the Tel-kinase- transformed Ba͞F3 cells, a collection of Ϸ80 tyrosine kinase inhibitors with known activities and selectivities was tested in dose–response format against the cell panel. For example, the Sugen c-Met inhibitor PHA-665752 inhibited the growth of Ba͞ F3͞Tel-Met with an IC50 of 80 nM and showed no toxicity toward the rest of the panel, in agreement with published results (19). Likewise, the kinase-insert domain receptor (KDR) inhibitor AAL993 (20) shows selective nanomolar potency against Ba͞F3͞ Fig. 1. The components of the ACP system. (A) Three custom environmen- Tel-KDR and the homologous VEGF receptor family member tally controlled (temperature, CO2, and humidity) 486-plate or flask capacity FLT4. Known BCR-ABL inhibitors, STI571 and AMN107, were incubators. (B) Combination flask to 1,536-well plate cell and reagent dis- also tested against the Ba͞F3 Tel-TK panel and a panel of recom- penser. (C) Control station consisting of a computer running custom schedul- binant enzymes. As shown in Fig. 2c, both STI571 and AMN107 ing software. (D) Perkin–Elmer ViewLux plate reader. (E) Olympus (Melville, inhibited Bcr-Abl, c-Kit, and PDGF-RA͞B with IC50s between 1 NY) inverted microscope with an automated stage and Molecular Devices and 100 nM as expected [data are shown as percent activity of the METAMORPH software.